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1. Reconstitution of circadian clock in synthetic cells reveals principles of timekeeping

   https://doi.org/10.1038/s41467-025-61844-5  

   Li, Alexander_Zhan_Tu; LiWang, Andy; Subramaniam, Anand_Bala (July 2025, Nature Communications)

   Abstract The cyanobacterial circadian clock maintains remarkable precision and synchrony, even in cells with femtoliter volumes. Here, we reconstitute the KaiABC post-translational oscillator (PTO) in giant unilamellar vesicles (GUVs) to investigate underlying mechanisms of this fidelity. We show that our encapsulation methodology replicates native protein variability. With long-term, single-vesicle tracking of circadian rhythms using fluorescent KaiB and confocal microscopy, we find that oscillator fidelity decreases with lower protein levels and smaller vesicle sizes. KaiB membrane association, observed in cyanobacteria, was recapitulated in GUV membranes. A mathematical model incorporating protein stoichiometry limitations suggests that high expression of PTO components and associated regulators (CikA and SasA) buffers stochastic variations in protein levels. Additionally, while the transcription-translation feedback loop contributes minimally to overall fidelity, it is essential for maintaining phase synchrony. These findings demonstrate synthetic cells capable of autonomous circadian rhythms and highlight a generalizable strategy for dissecting emergent biological behavior using minimal systems. 

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2. Time-course RNASeq of Camponotus floridanus forager and nurse ant brains indicate links between plasticity in the biological clock and behavioral division of labor

   https://doi.org/10.1186/s12864-021-08282-x  

   Das, Biplabendu; de Bekker, Charissa (January 2022, BMC Genomics)

   Abstract BackgroundCircadian clocks allow organisms to anticipate daily fluctuations in their environment by driving rhythms in physiology and behavior. Inter-organismal differences in daily rhythms, called chronotypes, exist and can shift with age. In ants, age, caste-related behavior and chronotype appear to be linked. Brood-tending nurse ants are usually younger individuals and show “around-the-clock” activity. With age or in the absence of brood, nurses transition into foraging ants that show daily rhythms in activity. Ants can adaptively shift between these behavioral castes and caste-associated chronotypes depending on social context. We investigated how changes in daily gene expression could be contributing to such behavioral plasticity inCamponotus floridanuscarpenter ants by combining time-course behavioral assays and RNA-Sequencing of forager and nurse brains. ResultsWe found that nurse brains have three times fewer 24 h oscillating genes than foragers. However, several hundred genes that oscillated every 24 h in forager brains showed robust 8 h oscillations in nurses, including the core clock genesPeriodandShaggy. These differentially rhythmic genes consisted of several components of the circadian entrainment and output pathway, including genes said to be involved in regulating insect locomotory behavior. We also found thatVitellogenin, known to regulate division of labor in social insects, showed robust 24 h oscillations in nurse brains but not in foragers. Finally, we found significant overlap between genes differentially expressed between the two ant castes and genes that show ultradian rhythms in daily expression. ConclusionThis study provides a first look at the chronobiological differences in gene expression between forager and nurse ant brains. This endeavor allowed us to identify a putative molecular mechanism underlying plastic timekeeping: several components of the ant circadian clock and its output can seemingly oscillate at different harmonics of the circadian rhythm. We propose that such chronobiological plasticity has evolved to allow for distinct regulatory networks that underlie behavioral castes, while supporting swift caste transitions in response to colony demands. Behavioral division of labor is common among social insects. The links between chronobiological and behavioral plasticity that we found inC. floridanus, thus, likely represent a more general phenomenon that warrants further investigation. 

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3. Connectomic analysis of the Drosophila lateral neuron clock cells reveals the synaptic basis of functional pacemaker classes

   https://doi.org/10.7554/eLife.79139  

   Shafer, Orie T; Gutierrez, Gabrielle J; Li, Kimberly; Mildenhall, Amber; Spira, Daphna; Marty, Jonathan; Lazar, Aurel A; Fernandez, Maria de (June 2022, eLife)

   Most organisms on Earth possess an internal timekeeping system which ensures that bodily processes such as sleep, wakefulness or digestion take place at the right time. These precise daily rhythms are kept in check by a master clock in the brain. There, thousands of neurons – some of which carrying an internal ‘molecular clock’ – connect to each other through structures known as synapses. Exactly how the resulting network is organised to support circadian timekeeping remains unclear. To explore this question, Shafer, Gutierrez et al. focused on fruit flies, as recent efforts have systematically mapped every neuron and synaptic connection in the brain of this model organism. Analysing available data from the hemibrain connectome project at Janelia revealed that that the neurons with the most important timekeeping roles were in fact forming the fewest synapses within the network. In addition, neurons without internal molecular clocks mediated strong synaptic connections between those that did, suggesting that ‘clockless’ cells still play an integral role in circadian timekeeping. With this research, Shafer, Gutierrez et al. provide unexpected insights into the organisation of the master body clock. Better understanding the networks that underpin circadian rhythms will help to grasp how and why these are disrupted in obesity, depression and Alzheimer’s disease. 

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4. Circadian Interactomics: How Research Into Protein-Protein Interactions Beyond the Core Clock Has Influenced the Model of Circadian Timekeeping

   https://doi.org/10.1177/07487304211014622  

   Mosier, Alexander_E; Hurley, Jennifer_M (May 2021, Journal of Biological Rhythms)

   The circadian clock is the broadly conserved, protein-based, timekeeping mechanism that synchronizes biology to the Earth’s 24-h light-dark cycle. Studies of the mechanisms of circadian timekeeping have placed great focus on the role that individual protein-protein interactions play in the creation of the timekeeping loop. However, research has shown that clock proteins most commonly act as part of large macromolecular protein complexes to facilitate circadian control over physiology. The formation of these complexes has led to the large-scale study of the proteins that comprise these complexes, termed here “circadian interactomics.” Circadian interactomic studies of the macromolecular protein complexes that comprise the circadian clock have uncovered many basic principles of circadian timekeeping as well as mechanisms of circadian control over cellular physiology. In this review, we examine the wealth of knowledge accumulated using circadian interactomics approaches to investigate the macromolecular complexes of the core circadian clock, including insights into the core mechanisms that impart circadian timing and the clock’s regulation of many physiological processes. We examine data acquired from the investigation of the macromolecular complexes centered on both the activating and repressing arm of the circadian clock and from many circadian model organisms. 

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5. The Drosophila circadian clock gene cycle controls the development of clock neurons

   https://doi.org/10.1371/journal.pgen.1011441  

   Biondi, Grace; McCormick, Gina; Fernandez, Maria P (October 2024, PLOS Genetics)

   Ewer, John (Ed.)

   Daily behavioral and physiological rhythms are controlled by the brain’s circadian timekeeping system, a synchronized network of neurons that maintains endogenous molecular oscillations. These oscillations are based on transcriptional feedback loops of clock genes, which inDrosophilainclude the transcriptional activatorsClock (Clk)andcycle (cyc). While the mechanisms underlying this molecular clock are very well characterized, the roles that the core clock genes play in neuronal physiology and development are much less understood. TheDrosophilatimekeeping center is composed of ~150 clock neurons, among which the four small ventral lateral neurons (sLN_vs) are the most dominant pacemakers under constant conditions. Here, we show that downregulating the clock genecycspecifically in thePdf-expressing neurons leads to decreased fasciculation both in larval and adult brains. This effect is due to a developmental role ofcyc, as both knocking downcycor expressing a dominant negative form ofcycexclusively during development lead to defasciculation phenotypes in adult clock neurons.Clkdownregulation also leads to developmental effects on sLNv morphology. Our results reveal a non-circadian role forcyc, shedding light on the additional functions of circadian clock genes in the development of the nervous system. 

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